Molecular cloning and functional expression of mannitol-1-phosphatase from the apicomplexan parasite Eimeria tenella.
A metabolic pathway responsible for the biosynthesis and utilization of mannitol is present in the seven species of Eimeria that infect chickens, but is not in the avian host. Mannitol-1-phosphatase (M1Pase), a key enzyme for mannitol biosynthesis, is a highly substrate-specific phosphatase and, accordingly, represents an attractive chemotherapeutic target. Amino acid sequence of tryptic peptides obtained from biochemically purified Eimeria tenella M1Pase was used to synthesize degenerate oligonucleotide hybridization probes. Using these reagents, a partial genomic clone and full-length cDNA clones have been isolated and characterized. The deduced amino acid sequence of E. tenella M1Pase shows limited overall homology to members of the phosphohistidine family of phosphatases. This limited homology to other histidine phosphatases does, however, include several conserved residues that have been shown to be essential for their catalytic activity. Kinetic parameters of recombinant M1Pase expressed in bacteria are essentially identical to those of the biochemically purified preparation from E. tenella. Moreover, recombinant M1Pase is subject to active site-directed, hydroxylamine-reversible inhibition by the histidine-selective acylating reagent diethyl pyrocarbonate. These results indicate the presence of an essential histidine residue(s) at the M1Pase active site, as predicted for a histidine phosphatase.
Liberator
P
P
Department of Parasite Biochemistry and Cell Biology, Merck Research Laboratories, Rahway, New Jersey 07065, USA. paul_liberator@merck.com
Anderson
J
J
Feiglin
M
M
Sardana
M
M
Griffin
P
P
Schmatz
D
D
Myers
R W
RW
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D016786:Eimeria tenella / Q000201:enzymology*; D015870:Gene Expression / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014357:Trypsin / Q000378:metabolism
D010446:Peptide Fragments; D011994:Recombinant Proteins; D010744:Phosphoric Monoester Hydrolases; C021543:mannitol-1-phosphatase; D014357:Trypsin; D004047:Diethyl Pyrocarbonate